Fluidic device for quantifying the dynamic permeability and hydraulic conductivitiy of living tissue layers

ABSTRACT

Systems and methods for measuring dynamic hydraulic conductivity and permeability associated with a cell layer are disclosed. Some systems include a microfluidic device, one or more working-fluid reservoirs, and one or more fluid-resistance element. The microfluidic device includes a first microchannel, a second microchannel, and a barrier therebetween. The barrier includes a cell layer adhered thereto. The working fluids are delivered to the microfluidic device. The fluid-resistance elements are coupled to one or more of the fluid paths and provide fluidic resistance to cause a pressure drop across the fluid-resistance elements. Mass transfer occurs between the first microchannel and the second microchannel, which is indicative of the hydraulic conductivity and/or dynamic permeability associated with the cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2016/021025, filed Mar. 4, 2016, which claims the benefit of U.S.Provisional Application No. 62/128,383, filed Mar. 4, 2015, each ofwhich is herein incorporated by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant no.W911NF-12-2-0036 awarded by U.S. Department of Defense, AdvancedResearch Projects Agency. The government has certain rights in theinvention.

TECHNICAL FIELD

The present invention relates to cell culture systems and fluidicsystems. More specifically, the invention relates to systems thatprovide for improved characterization of the permeability and hydraulicconductivity of dynamic environments, including systems havingbiological cell layers.

BACKGROUND

Typical laboratory set-ups for measuring hydraulic conductivity of celllayers are generally based on a modified Transwell assay where thetissue is cultured on a semi-permeable substrate or the membrane of aTranswell insert. Fluid is then supplied to the well and a pressuregradient is created between the two sides of the semi-permeablesubstrate. The fluid is generally allowed to only flow perpendicularlyto the surface of the Transwell membrane. Monitoring the fluid flow ratefrom the insert compartment to the bottom compartment can be used toquantify a static hydraulic conductivity through the tissue. Thepressure gradient can be induced by establishing an air-tight connectionbetween the insert and an open reservoir. When quantifying the flow ratein the system (e.g., by collecting outflow or by tracking a bubblemoving through capillary tubing), the hydraulic conductivity of thecultured tissue in a static system can be determined. Unfortunately,in-vivo systems are more dynamic and, thus, not accurately modeled bythese set-ups.

Combining the set-ups based on a Transwell membrane with a spinning discor spinning cone rheometer may allow the tissue be subjected to someshear stresses. Studies using such a combined set-up indicate thatsubjecting the tissue to shear stresses has an important modulatoryeffect on hydraulic conductivity of tissue, such as endothelial tissue.However, the combined system prevents certain beneficial features, suchas monitoring the cells during the experiment and providing forprolonged experimentation. Moreover, these combined set-ups increasecost and complexity of the experiment due to the need for expensiveequipment with a number of moving parts. Most importantly, combinedset-ups, such as spinning disk or spinning cone rheometers, producecircular patterns of shear stress. These circular flow patterns do notprovide accurate simulation of biological conditions.

Another disadvantage of prior art static and combined set-ups is thatthey do not incorporate ways to dynamically alter the flow rates andpressure gradients in the system. These parameters can change over timein real-life tissues, and these changes can have important roles inphysiological processes that involve changes in hydraulic conductivity.For example, when a blood vessel ruptures, the hydraulic conductivity ofthe vessel wall is increased for only a brief time. Within minutes, thelocal pressure gradient is decreased by vasospasm and the hydraulicconductivity of the ruptured wall is lowered by blood clotting.

Additionally, the permeability of biological tissues for specificsubstances or particles is an important parameter in fields like drugdiscovery, toxicology, and physiology. For example, the extent that asubstance can permeate specific tissues determines important aspects ofthe substance's pharmacokinetics and toxic risk profile. Additionally,the proper functioning of tissues can be probed by testing theirpermeability to standard tracer molecules. These tracer molecules caninclude relatively inert compounds such as dextran, inulin, andpolyethylene glycol, or biologically active compounds such as glucose.

Typical laboratory setups for measuring permeability of cell layers aregenerally based on modified Transwell assays where tissue is cultured ona semi-permeable substrate or the membrane of a Transwell insert. Theinsert is filled with medium including the substance of interest andthen placed in a well that has been filled with control medium. Theheight of the media in the insert and the well are matched to preventconvective transport through the tissue driven by hydrostatic pressure.By repetitively taking samples from the well, the rate at which asubstance diffuses through the tissue can be determined. Afternormalizing for the concentration gradient and the surface area, thisrate of diffusion is defined as the permeability coefficient of thatparticular substance for that particular tissue.

Attempts have been made to produce dynamic assays for measuringpermeability of cell layers through use of microfluidic devices. Thesedynamic assays rely on the same principles as described above byemploying two compartments separated by a semi-permeable barrier that iscovered by tissue. However, the media in the compartments is movingthrough the microfluidic device in the dynamic version of the assay.This flow of media is needed to maintain a stable concentration gradientover the cultured tissue. Unfortunately, the volumes of the microfluidiccompartments are so low that even small absolute amounts of mediatransferred through the tissue can dramatically affect the measurementof concentrations in the system.

A major challenge when performing dynamic permeability assays inmicrofluidic systems is to avoid convective transport between the twocompartments. Even small differences in the fluidic resistance betweenthe two compartments can drive a cross-flow from one compartment to theother. Differences in fluidic resistance may be a result of the designof the microfluidic device or experimental setup, such as differenthydraulic diameters for the two compartments, different downstreamlengths of the two compartments, or inherent differences due tomanufacturing tolerances. Further, differences in fluidic resistance canalso arise during operation of the systems such as when a channel isblocked with small pieces of dirt, clumps of tissue, or small airbubbles. These small differences make it very difficult, if notimpossible, to standardize the results of the assay because transport isdriven by both diffusion and convection. This also makes it difficult,if not impossible, to compare the results from the dynamic assay withresults from conventional Transwell studies, or to model whole tissuesand organs in vivo.

The present invention solves many of the problems associated with theprior art systems by providing new systems and methods for quantifyingthe dynamic hydraulic conductivity and the dynamic permeability of celllayers by use of fluidic and microfluidic devices having the cell layerslocated therein.

SUMMARY

According to aspects of the present invention, a system for measuringdynamic hydraulic conductivity associated with a cell layer includes amicrofluidic device, a working-fluid reservoir, a first fluid line, aflow-determining element, and a second fluid line. The microfluidicdevice includes a first microchannel, a second microchannel, and abarrier located at an interface region between the first microchanneland the second microchannel. The barrier includes a first side facingtoward the first microchannel and a second side facing toward the secondmicrochannel. The first side has the cell layer adhered thereto. Theworking-fluid reservoir includes a working fluid that is delivered tothe first microchannel. The first fluid line is for delivering a firstportion of a working fluid from the first microchannel to a first fluidreservoir. The flow-determining element is coupled to the first fluidline and is configured to determine flow of fluid therethrough. Thesecond fluid line is for delivering a second portion of the workingfluid from the second microchannel to a second fluid reservoir. Thesecond portion of the working fluid moves from the first microchannel tothe second microchannel through the cell layer and is indicative of thedynamic hydraulic conductivity associated with the cells.

According to further aspects of the present invention, a method formeasuring dynamic hydraulic conductivity associated with cells includesflowing a working fluid through a first microchannel, applying a firstpressure to the working fluid in the first microchannel, collecting afirst portion of the working fluid exiting the first microchannel,collecting the second portion of the working fluid exiting the secondmicrochannel, and calculating a dynamic hydraulic conductivity of thelayer of cells. The working fluid is flowed through the firstmicrochannel at a first flow rate along a layer of cells. The layer ofcells is disposed on a barrier. The first flow rate causes a first shearstress on the layer of cells. The first pressure is applied to theworking fluid in the first microchannel to cause a second portion of theworking fluid to travel to a second microchannel through the layer ofcells and the barrier. The calculating of the dynamic hydraulicconductivity of the layer of cells is based on the first pressure andthe second portion.

According to still further aspects of the present invention, a methodfor measuring dynamic hydraulic conductivity associated with cellsincludes moving a working fluid through a first microchannel of amicrofluidic device, measuring a first portion of the working fluid thatexits the first microchannel, measuring a second portion of the workingfluid that migrates through the cell layer and a barrier and exits asecond microchannel of the microfluidic device, and determining thedynamic hydraulic conductivity of the cells based on at least one of thefirst portion and the second portion of the working fluids.

According to yet further aspects of the present invention, a system formeasuring dynamic hydraulic conductivity associated with cells includesa microfluidic device, a first fluid path, and a second fluid path. Themicrofluidic device has a first microchannel, a second microchannel, anda barrier located at an interface region between the first microchanneland the second microchannel. The barrier includes a first side facingtoward the first microchannel and a second side facing toward the secondmicrochannel. At least one of the first side and the second side has acell layer adhered thereto. The first fluid path is associated with thefirst microchannel. The first fluid path is for delivering a fluid toand from the first microchannel. The first fluid path includes aflow-determining element downstream from the first microchannel tomaintain a substantially constant fluid pressure along the cell layeradhered to the barrier. The second fluid path is associated with thesecond microchannel. The second fluid path is for delivering, from thesecond microchannel, the fluid that has migrated through the barrier andthe cell layer. The flow rate of the fluid that is delivered from thesecond microchannel is indicative of the dynamic hydraulic conductivityassociated with the cell layer.

According to still yet further aspects of the present invention, asystem for measuring dynamic permeability of a cell layer includes amicrofluidic device, a first working-fluid reservoir, a first fluidline, a first fluid-resistance element, a second working-fluidreservoir, a second fluid line, and a second fluid-resistance element.The microfluidic device includes a first microchannel, a secondmicrochannel, and a barrier located at an interface region between thefirst microchannel and the second microchannel. The barrier includes afirst side facing toward the first microchannel and a second side facingtoward the second microchannel. The first side includes the cell layeradhered thereto. The first working-fluid reservoir contains a firstworking fluid that is delivered to the first microchannel. The firstfluid line delivers fluid from the first microchannel to a firstoutput-fluid reservoir. The first fluid-resistance element is coupled tothe first fluid line. The first fluid-resistance element has a firstfluidic resistance that causes a pressure drop across the firstfluid-resistance element. The second working-fluid reservoir contains asecond working fluid that is delivered to the second microchannel. Thesecond fluid line delivers fluid from the second microchannel to asecond output-fluid reservoir. The second fluid-resistance element iscoupled to the second fluid line. The second fluid-resistance elementhas a second fluidic resistance that causes a pressure drop across thesecond fluid-resistance element. The first fluidic resistance and thesecond fluidic resistance are substantially larger than the nominalresistances of the first and the second microchannel, respectively, soas to inhibit convective flow between the first working fluid and thesecond working fluid through the barrier and the cell layer, therebyallowing measurement of dynamic permeability through the cell layer.

According to additional aspects of the present invention, a method formeasuring dynamic permeability of a cell layer includes the acts ofmoving a first working fluid through a first fluid path, moving a secondworking fluid through a second fluid path, and measuring an analyte inthe second working fluid. The first fluid path includes a firstmicrochannel of a microfluidic device and a first fluid-resistanceelement. The microfluidic device further includes a second microchanneland a barrier located at an interface region between the firstmicrochannel and the second microchannel. The barrier includes a firstside facing toward the first microchannel and has the cells adheredthereto. The first fluid-resistance element has a first fluidicresistance that causes a pressure drop across the first fluid-resistanceelement. The first working fluid includes the analyte. The second fluidpath includes the second microchannel of the microfluidic device and asecond fluid-resistance element. The second fluid-resistance element hasa second fluidic resistance that causes a pressure drop across thesecond fluid-resistance element. The measuring the analyte in the secondworking fluid occurs after the second working fluid has passed throughthe second microchannel. The measured analyte is indicative of thedynamic permeability of the cell layer.

According to yet additional aspects of the present invention, a systemfor measuring dynamic permeability of a cell layer includes amicrofluidic device, a first working-fluid reservoir, a first fluidline, a first fluid-resistance element, a second working-fluidreservoir, a second fluid line, and a second fluid-resistance element.The microfluidic device includes a first microchannel, a secondmicrochannel, and a barrier located at an interface region between thefirst microchannel and the second microchannel. The barrier includes afirst side facing toward the first microchannel and a second side facingtoward the second microchannel. The first side has the cell layeradhered thereto. The first working-fluid reservoir contains a firstworking fluid that is delivered to the first microchannel. The firstfluid line delivers fluid away from the first microchannel. The firstfluid-resistance element is coupled to the first fluid line. The firstfluid-resistance element includes a first fluidic resistance that issubstantially larger than the nominal resistance of the firstmicrochannel. The second working-fluid reservoir contains a secondworking fluid that is delivered to the second microchannel. The secondfluid line delivers fluid away from the second microchannel. The secondfluid-resistance element is coupled to the second fluid line. The secondfluid-resistance element has a second fluidic resistance that issubstantially larger than the nominal resistance of the secondmicrochannel. The first fluidic resistance and the second fluidicresistance create a negligible pressure drop across the barrier and thecell layer while the first working fluid and the second working fluidflow through the system, thereby allowing the measurement of dynamicpermeability through the barrier and the cell layer.

According to aspects of the present invention, a system for measuringdynamic hydraulic conductivity associated with a cell layer includes afluidic device, a working-fluid reservoir, a first fluid line, aflow-determining element, and a second fluid line. The fluidic deviceincludes a first channel, a second channel, and a barrier located at aninterface region between the first channel and the second channel. Thebarrier includes a first side facing toward the first channel and asecond side facing toward the second channel. The first side has thecell layer adhered thereto. The working-fluid reservoir includes aworking fluid that is delivered to the first channel. The first fluidline is for delivering a first portion of a working fluid from the firstchannel to a first fluid reservoir. The flow-determining element iscoupled to the first fluid line and is configured to determine flow offluid therethrough. The second fluid line is for delivering a secondportion of the working fluid from the second channel to a second fluidreservoir. The second portion of the working fluid moves from the firstchannel to the second channel through the cell layer and is indicativeof the dynamic hydraulic conductivity associated with the cells.

According to further aspects of the present invention, a method formeasuring dynamic hydraulic conductivity associated with cells includesflowing a working fluid through a first channel, applying a firstpressure to the working fluid in the first channel, collecting a firstportion of the working fluid exiting the first channel, collecting thesecond portion of the working fluid exiting the second channel, andcalculating a dynamic hydraulic conductivity of the layer of cells. Theworking fluid is flowed through the first channel at a first flow ratealong a layer of cells. The layer of cells is disposed on a barrier. Thefirst flow rate causes a first shear stress on the layer of cells. Thefirst pressure is applied to the working fluid in the first channel tocause a second portion of the working fluid to travel to a secondchannel through the layer of cells and the barrier. The calculating ofthe dynamic hydraulic conductivity of the layer of cells is based on thefirst pressure and the second portion.

According to still further aspects of the present invention, a methodfor measuring dynamic hydraulic conductivity associated with cellsincludes moving a working fluid through a first channel of a fluidicdevice, measuring a first portion of the working fluid that exits thefirst channel, measuring a second portion of the working fluid thatmigrates through the cell layer and a barrier and exits a second channelof the fluidic device, and determining the dynamic hydraulicconductivity of the cells based on at least one of the first portion andthe second portion of the working fluids.

According to yet further aspects of the present invention, a system formeasuring dynamic hydraulic conductivity associated with cells includesa fluidic device, a first fluid path, and a second fluid path. Thefluidic device has a first channel, a second channel, and a barrierlocated at an interface region between the first channel and the secondchannel. The barrier includes a first side facing toward the firstchannel and a second side facing toward the second channel. At least oneof the first side and the second side has a cell layer adhered thereto.The first fluid path is associated with the first channel. The firstfluid path is for delivering a fluid to and from the first channel. Thefirst fluid path includes a flow-determining element downstream from thefirst channel to maintain a substantially constant fluid pressure alongthe cell layer adhered to the barrier. The second fluid path isassociated with the second channel. The second fluid path is fordelivering, from the second channel, the fluid that has migrated throughthe barrier and the cell layer. The flow rate of the fluid that isdelivered from the second channel is indicative of the dynamic hydraulicconductivity associated with the cell layer.

According to still yet further aspects of the present invention, asystem for measuring dynamic permeability of a cell layer includes afluidic device, a first working-fluid reservoir, a first fluid line, afirst fluid-resistance element, a second working-fluid reservoir, asecond fluid line, and a second fluid-resistance element. The fluidicdevice includes a first channel, a second channel, and a barrier locatedat an interface region between the first channel and the second channel.The barrier includes a first side facing toward the first channel and asecond side facing toward the second channel. The first side includesthe cell layer adhered thereto. The first working-fluid reservoircontains a first working fluid that is delivered to the first channel.The first fluid line delivers fluid from the first channel to a firstoutput-fluid reservoir. The first fluid-resistance element is coupled tothe first fluid line. The first fluid-resistance element has a firstfluidic resistance that causes a pressure drop across the firstfluid-resistance element. The second working-fluid reservoir contains asecond working fluid that is delivered to the second channel. The secondfluid line delivers fluid from the second channel to a secondoutput-fluid reservoir. The second fluid-resistance element is coupledto the second fluid line. The second fluid-resistance element has asecond fluidic resistance that causes a pressure drop across the secondfluid-resistance element. The first fluidic resistance and the secondfluidic resistance are substantially larger than the nominal resistancesof the first and the second channel, respectively, so as to inhibitconvective flow between the first working fluid and the second workingfluid through the barrier and the cell layer, thereby allowingmeasurement of dynamic permeability through the cell layer.

According to additional aspects of the present invention, a method formeasuring dynamic permeability of a cell layer includes the acts ofmoving a first working fluid through a first fluid path, moving a secondworking fluid through a second fluid path, and measuring an analyte inthe second working fluid. The first fluid path includes a first channelof a fluidic device and a first fluid-resistance element. The fluidicdevice further includes a second channel and a barrier located at aninterface region between the first channel and the second channel. Thebarrier includes a first side facing toward the first channel and hasthe cells adhered thereto. The first fluid-resistance element has afirst fluidic resistance that causes a pressure drop across the firstfluid-resistance element. The first working fluid includes the analyte.The second fluid path includes the second channel of the fluidic deviceand a second fluid-resistance element. The second fluid-resistanceelement has a second fluidic resistance that causes a pressure dropacross the second fluid-resistance element. The measuring the analyte inthe second working fluid occurs after the second working fluid haspassed through the second channel. The measured analyte is indicative ofthe dynamic permeability of the cell layer.

According to yet additional aspects of the present invention, a systemfor measuring dynamic permeability of a cell layer includes a fluidicdevice, a first working-fluid reservoir, a first fluid line, a firstfluid-resistance element, a second working-fluid reservoir, a secondfluid line, and a second fluid-resistance element. The fluidic deviceincludes a first channel, a second channel, and a barrier located at aninterface region between the first channel and the second channel. Thebarrier includes a first side facing toward the first channel and asecond side facing toward the second channel. The first side has thecell layer adhered thereto. The first working-fluid reservoir contains afirst working fluid that is delivered to the first channel. The firstfluid line delivers fluid away from the first channel. The firstfluid-resistance element is coupled to the first fluid line. The firstfluid-resistance element includes a first fluidic resistance that issubstantially larger than the nominal resistance of the first channel.The second working-fluid reservoir contains a second working fluid thatis delivered to the second channel. The second fluid line delivers fluidaway from the second channel. The second fluid-resistance element iscoupled to the second fluid line. The second fluid-resistance elementhas a second fluidic resistance that is substantially larger than thenominal resistance of the second channel. The first fluidic resistanceand the second fluidic resistance create a negligible pressure dropacross the barrier and the cell layer while the first working fluid andthe second working fluid flow through the system, thereby allowing themeasurement of dynamic permeability through the barrier and the celllayer.

These and other capabilities of the invention, along with the inventionitself, will be more fully understood after a review of the followingfigures, detailed description, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

While the invention is susceptible to various modifications andalternative forms, specific embodiments have been shown by way ofexample in the drawings and will be described in detail herein. Itshould be understood, however, that the invention is not intended to belimited to the particular forms disclosed. Rather, the invention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the appended claims.

FIG. 1 illustrates an exemplary organ-on-chip device that may be usedwith systems of the present invention.

FIG. 2 is a cross-section of the organ-on-chip device taken along line2-2 of FIG. 1, illustrating the first and second microchannel of theorgan-on-chip device.

FIG. 3 is a cross-section of the organ-on-chip device taken along line3-3 of FIG. 2, illustrating fluid flow between the first microchanneland the second microchannel of the organ-on-chip device of FIG. 1.

FIG. 4 illustrates a schematic representation of a system forquantifying the dynamic hydraulic conductivity of biological celllayers, according to aspects of the present invention.

FIG. 5 illustrates a schematic representation of a system forquantifying the dynamic hydraulic conductivity of biological celllayers, according to further aspects of the present invention.

FIG. 6 illustrates a schematic representation of a system forquantifying the dynamic permeability of biological cell layers,according to aspects of the present invention.

FIG. 7 illustrates a flow schematic of the system of FIG. 6.

FIG. 8 illustrates a schematic representation of a system forquantifying fluid transport across biological cell layers, according toaspects of the present invention.

DETAILED DESCRIPTION

While this invention is susceptible of embodiment in many differentforms, there is shown in the drawings and will herein be described indetail preferred embodiments of the invention with the understandingthat the present disclosure is to be considered as an exemplification ofthe principles of the invention and is not intended to limit the broadaspect of the invention to the embodiments illustrated.

The bulk flow or convective transfer of fluids between differentcompartments in the human body is determined by pressure gradients andthe hydraulic conductivity of the tissue between the compartments.Hydraulic conductivity is a quantification of the rate at which a fluidsuch as water flows through a layer of tissue. Hydraulic conductivity isgiven as the volumetric flow rate divided by the area and pressuredifference. Some physiological examples of fluid flow between differentcompartments in the body are the filtration of blood in the capillaries,the interstitial flow through three-dimensional compartments likecartilage and other tissues, the filtration of blood in the glomeruli ofthe kidney, and the filtration of blood in the choroid plexus and thecerebral capillaries to generate cerebrospinal fluid.

Diffusive or quasi-diffusive transfer of fluids or components of fluidsbetween different compartments in the human body is determined by thepermeability of the tissues. The permeability of the cell layer differsdepending on a variety of factors such as the analyte being studied, theconcentration gradient across the cell layer, the surface area of thecell layer, the shear forces on the surface of the cell layer, the stateof the cell layer, the capability of the cell layer to activelytransport or pump back the analyte, etc. Some in-vivo examples ofpermeability of the cell layer include transfer and accumulation oftoxic compounds, pharmacological compounds, physiological compounds, orthe like.

Some, if not all, tissues can sense fluid flow running through or alongthem and can have a physiologically relevant response to it.Additionally, alterations in the hydraulic conductivity of tissues areinvolved in many pathological states. For example, inflammation intissues can lead to endothelial or epithelial increases in hydraulicconductivity. These changes are intimately linked to pathologicalprocesses like hemorrhage and proteinuria. In three-dimensional tissueslike cartilage and bone, inflammation can lead to changes in thehydraulic conductivity, which can cause, for example, joint stiffening.

In order to model physiological and pathological processes, and todetermine the hydraulic conductivity and permeability of specifictissues and cell layers, in-vitro modeling is an indispensable tool. Thepresent invention relates to fluidic devices, systems, and methods thatprovide for determining hydraulic conductivity and permeability oftissues under dynamic conditions. Fluidic devices, systems, and methodsin accord with the present invention allow for many benefits overexisting systems, such as more accurate modeling of physical systems.More specifically, fluidic devices, systems, and methods disclosedherein can subject tissues and cell cultures to dynamic conditions thatmore closely simulate living systems. For example, fluidic devices,systems, and methods as disclosed herein can produce non-circular shearstresses across exposed surfaces of the tissues and cultured celllayers, and can produce physiologically relevant flow patterns such aslaminar flow, turbulent flow, pulsating flow, and the like. Theincorporation of physiologically relevant flow over the surface of acell layer while monitoring its hydraulic conductivity and permeabilityincreases the realism, versatility, and physiological relevance of theset-up. Moreover, fluidic devices, systems, and methods as disclosedherein can provide for monitoring dynamic hydraulic conductivity anddynamic permeability changes in real-time.

Further, embodiments of the present invention provide for increasedaccuracy of physiological models because conditions such as flow rates,pressure gradients, and supplied analyte concentrations can be variedwhile using the same tissue or cultured cell layer without disturbingthe cells, thus providing more accurate characterization by inhibitingthe effects of batch-to-batch inconsistency. The present invention alsoprovides for altering pressure gradients, flow rates, and/or suppliedanalyte concentrations in a simple, straightforward way by simplychanging or modifying one or more downstream flow-determining elements,as will be described in more detail below.

Yet further, embodiments of the present invention provide for systemsand methods that are more robust by reducing or eliminating the need formoving parts, and allow for use of various analytical or image-gatheringtechniques, such as spectroscopy and microscopy on cells in real time asthe experiment proceeds. Additionally, the present invention may allowfor hydraulic conductivity of the same cells to be measured over thecourse of hours, days, weeks, etc.

Still yet further, embodiments of the present invention provide forsystems and methods that are more robust and allow for long-termconsistency in the testing procedure. For example, fluidic-resistanceelements can be used to inhibit flow differences caused by blockages,such as small pieces of dirt, clumps of tissue, or small air bubbles.This allows for precise measurement of permeability coefficients even insystems that have an inherent or unwanted difference in fluidicresistance between the two fluidic compartments. Further, it allows fordata collection over a longer period of where the occurrence of suchblockages is generally unavoidable.

The functionality of cells and tissue types (and even organs) can beimplemented in one or more microfluidic devices or “chips” that enableresearchers to study these cells and tissue types outside of the bodywhile mimicking much of the stimuli and environment that the tissue isexposed to in-vivo. It can also be desirable to implement thesemicrofluidic devices into interconnected components that can simulategroups of organs or tissue systems. Preferably, the microfluidic devicescan be easily inserted and removed from an underlying fluidic systemthat connects to these devices in order to vary the simulated in-vivoconditions and organ systems.

FIGS. 1 and 2 illustrate one type of an organ-on-chip (“OOC”) device 10.The 00C device 10 includes a body 12 that is typically made of apolymeric material. The body 12 includes a first fluid inlet 14 a and afirst fluid outlet 14 b. The body 12 further includes a second fluidinlet 16 a and a second fluid outlet 16 b. The first fluid inlet 14 aand the first fluid outlet 14 b allow fluid flow through a firstmicrochannel 24. The second fluid inlet 16 a and the second fluid outlet16 b allow fluid flow through a second microchannel 26. The firstmicrochannel 24 is separated from the second microchannel 26 by abarrier 30. The barrier 30 may be any suitable semi-permeable barrierthat permits migration of cells, particulates, media, proteins, and/orchemicals between the first microchannel 24 and the second microchannel26. For example, the barrier 30 can include gels, layers of differenttissue, arrays of micro-pillars, membranes, combinations thereof, andthe like. Depending on the application, the barrier 30 may have openingsor pores to permit the migration of cells, particulates, media,proteins, and/or chemicals between the first microchannel 24 and thesecond microchannel 26. In some preferred embodiments, the barrier 30 isa porous membrane that includes a cell layer 34 disposed on at least afirst surface of the membrane.

While the illustrated embodiment includes only a single cell layer 34,the barrier 30 may include more than a single cell layer 34 disposedthereon. For example, the barrier 30 can include the cell layer 34disposed within the first microchannel 24, the second microchannel 26,or each of the first and second microchannels 24, 26. Additionally oralternatively, the barrier 30 can include a first cell layer 34 disposedwithin the first microchannel 24 and a second cell layer within thesecond microchannel 26. Additionally or alternatively, the barrier 30can include a first cell layer 34 and a second cell layer disposedwithin the first microchannel 24, the second microchannel 26, or each ofthe first and second microchannels 24, 26. Extracellular matrix gels canbe used in addition to or instead of the cell layers. Beneficially,these various combinations provide for in-vitro modeling of variouscells, tissues, and organs including three-dimensional structures andtissue-tissue interfaces such as brain astrocytes, kidney glomuralarepithelial cells, etc. In one embodiment of the OOC device 10, the firstand second microchannels 24, 26 generally have a length of less thanabout 2 cm, a height of less than 200 and a width of less than 400 μm.More details on the OOC device 10 can be found in, for example, U.S.Pat. No. 8,647,861, which is owned by the assignee of the presentapplication and is incorporated by reference in its entirety.

The OOC device 10 is configured to simulate a biological functionassociated with cells, such as simulated organs, tissues, etc. One ormore properties of a working fluid may change as the working fluid ispassed through the microchannels 24, 26 of the OOC device 10, producingan effluent. As such, the effluent is still a part of the working fluid,but its properties and/or constituents may change when passing throughthe OOC device 10.

The OOC device 10 preferably includes an optical window that permitsviewing of the fluid as it moves across the cell layer 34 and thebarrier 30. Various image-gathering techniques, such as spectroscopy andmicroscopy, can be used to quantify and evaluate the fluid flow oranalyte flow through the cell layer 34 and the effect of shear on thecell layer 34 that is caused by different flow rates across the celllayer 34.

FIG. 3 schematically illustrates a cross-sectional view of the OOCdevice 10 across the length of the first and second microchannels 24, 26along line 3-3 in FIG. 2. The barrier 30 includes pores 31, which canhave various dimensions based on the barrier 30 that is chosen. In theillustrated example, a cell layer 34 is disposed within the firstmicrochannel 24 and on the first upper surface of the barrier 30. Fluidenters the first microchannel 24 and flows from the inlet toward theoutlet of the first microchannel 24. As the fluid flows from the inlettoward the outlet of the first microchannel 24, contact between thefluid and the surface of the cells 34 exerts a shear stress on the cells34. This shear stress can deform the individual cells 34, or affectother changes in the physical or biological properties of the cells 34.

In some embodiments for quantifying the hydraulic conductivity ofbiological cell layers, there is no fluid that enters the second fluidinlet 16 a of the second microchannel 26 such that the only fluidentering the OOC device 10 is from the first fluid inlet 14 a. In someembodiments, the second microchannel 26 is filled with the fluid at theinitiation of the hydraulic testing such that the hydraulic conductivitycan be measured contemporaneously with the start of the experiment. Inother embodiments, the second microchannel 26 is devoid of fluids at theinitiation of the hydraulic testing, and the fluid collected is onlyfluid that has traversed the barrier 30 during testing. Beneficially,these embodiments provide for simplified measurements of hydraulicconductivity because fewer variables are involved in the test (e.g.,instantaneous flow rate through the microchannels, instantaneouspressure within the microchannels, etc.) to determine the hydraulicconductivity.

In some embodiments for quantifying the hydraulic conductivity ofbiological cell layers, fluid is supplied to the second fluid inlet 16 ausing a constant-flow element. The constant flow element can be, forexample, a volumetric pump or a reservoir having fluid at a generallyconstant height above the OOC device 10. The constant-flow element isconfigured to provide a low flow rate to the second fluid inlet 16 asuch that no significant pressure is generated in the lower channel andthe pressure gradient between the first microchannel 24 and the secondmicrochannel 26 is maintained. The hydraulic conductivity can then bequantified by measuring the collected fluid and analyzing the increasein flow rate out of the second microchannel 26.

In some embodiments for testing the hydraulic conductivity of a celllayer, the fluid flows through the first microchannel 24 at a higherpressure than fluid in the second microchannel 26. This creates apressure gradient ΔP across the barrier 30. If the pressure issufficiently large, the fluid will travel from the first microchannel 24to the second microchannel 26 through the cell layer 34 and the pores 31of the barrier 30, shown by arrows 32 c. The fluid that has traversedthe cell layer 34 and the barrier 30, exits the second microchannel witha flow rate Q_(t) as shown by arrow 32 b. Because no other fluid hasentered the system, the sum of the flow rates out of the flow rates outof the first microchannel Q₁ and the second microchannel Q_(t) willequal the flow rate into the first microchannel Q₁. That is,Q₁=Q₂+Q_(t).

In some embodiments for testing the hydraulic conductivity of the celllayer, fluid flows through the first microchannel 24 at the samepressure as fluid flowing through the second microchannel 26. Thisinhibits convective flow across the cell layer. However, the cell layermay still transfer fluid between the first microchannel 24 and thesecond microchannel 26 through active transport processes (e.g.,hydraulic pumping). Accordingly, this transport can still be measuredunder dynamic conditions, and the hydraulic conductivity due tohydraulic pumping can be measured at a variety of flow rates.

The hydraulic conductivity K of the cell layer 34 and the barrier 30 ata predetermined flow rate Q₁ of media input to the first microchannel 24can be quantified by normalizing for the area A of the cell layer 34using the following equation:

$K = \frac{Q_{T}}{A\; \Delta \; P}$

Beneficially, the hydraulic conductivity of individual cell layers K_(L)can be determined by, for example, accounting for the hydraulicconductivity of the barrier K_(M). In other words, by knowing thehydraulic conductivity K of the system via the equation above and thehydraulic conductivity of the barrier K_(M), the hydraulic conductivityof individual cell layers K_(L) can be derived. Moreover, the hydraulicconductivity of the barrier K_(M) or fluidic resistance of the barriercan change dependent on the working fluid supplied. Alternatively, itmay be possible to select a barrier 30 that is sufficiently porous so asto generally not inhibit flow of media between the first microchannel 24and the second microchannel 26 relative to the cell layer 34. Similarly,if multiple cell layers 34 or multiple types of cells are co-culturedwithin the 00C device 10, the conductivity of each layer or cell typecan be determined by accounting for the known hydraulic conductivitiesof the other cell layers 34 or cell types.

Additionally, hydraulic conductivity as a function of shear forcesapplied to the cells can be determined. For example, hydraulicconductivity may increase in a non-linear fashion, or may increase instep fashion based on applied shear. For example, cells, such a vascularendothelial cells, exhibit a threshold-based response where conductivityof the cell layer is increased in response to a step-increase in luminalshear.

As discussed in more detail below, systems and methods in accord withthe present invention can provide for independent control of the flowrate Q₁ into the first microchannel 24 and the pressure differential ΔPbetween the first and the second microchannels 24, 26. Beneficially,these systems provide for more accurate determinations of the in-vivohydraulic conductivity of individual cell layers.

Referring now to FIG. 4, a schematic representation of a system 100 forquantifying the hydraulic conductivity of biological cell layers 34 isshown according to aspects of the present invention. The system 100includes a working-fluid reservoir 102, an OOC device 10, a flowdetermining element such as fluid-resistance element 104, a firstoutput-fluid reservoir 106 a, and a second output-fluid reservoir 106 b.The first input 14 a (FIG. 1) of the first microchannel 24 of the OOCdevice 10 is fluidically coupled to the working-fluid reservoir 102. Thefirst output 14 b (FIG. 1) of the first microchannel 24 of the OOCdevice 10 is fluidically coupled to the first output-fluid reservoir 106a. The second input 16 a (FIG. 1) for the second microchannel 26 of theOOC device 10 is plugged by stopper 116 to inhibit flow through thesecond microchannel 26. The second microchannel 26 of the OOC device 10is fluidically coupled to the second output-fluid reservoir 106 b.

The working-fluid reservoir 102 contains a working fluid 108 to bepassed through the system 100. In some embodiments, the working fluid108 is water to determine a baseline or normalized hydraulicconductivity value for the cells or tissue that is a function of water.In some embodiments, the working fluid 108 may be a liquid mediumincluding suspensions or mixtures of cells, particulates, proteins,chemicals, combinations thereof, or the like. In some embodiments, theworking fluid 108 may expose the cells of the OOC device 10 to acontaminant, pollutant, or pharmaceutical to determine the how the cellsreact to such exposure. The working-fluid reservoir 102 is coupled tothe first microchannel 24 of the OOC device 10 using input line 110.

The first output-fluid reservoir 106 a is coupled to the output of thefirst microchannel 24 of the OOC device 10 using a first output line 112a. The first output-fluid reservoir 106 a collects a first output fluid114 a that has passed through the first microchannel 24 of the OOCdevice 10. The second output-fluid reservoir 106 b is coupled to theoutput of the second microchannel 26 of the OOC device 10 using a secondoutput line 112 b. The second output-fluid reservoir 106 b collects asecond output fluid 114 b that has passed through the secondmicrochannel 26 of the OOC device 10 by migration through the barrier 30having cells thereon. As will be described in further detail below, theoutput-fluid reservoirs 106 a, 106 b can be used to monitor the flowrate through each microchannel 24, 26 and can allow for sampling of thefluid for chemical analysis, molecular analysis, cellular analysis,combinations thereof, and the like.

The flow-determining element is coupled to a fluid path such as thefluid path and associated with the first microchannel 24. Theflow-determining element is configured to determine flow of fluidthrough the fluid path. Beneficially, use of the flow-determiningelement provides for modularity of the system because flow through thefluid path can be controlled to accommodate differing operatingconditions of the OOC device 10, working-fluid reservoir 102, andoutput-fluid reservoirs 106 a, 106 b. In some embodiments, theflow-determining element is a pump such as a syringe pump. The pump canbe disposed downstream of the first microchannel 24 to determine flow offluid through the first microchannel 24. In some aspects, the syringepump draws in fluid from the fluid path to create a generally constantflow of fluid. In some aspects, the syringe pump is coupled to anoutput-fluid reservoir 106 a,b and draws in gas from the output-fluidreservoir 106 a,b. The drawing in of gas thereby draws fluid from thefluid path into the output-fluid reservoir 106 a,b. In some embodiments,the flow-determining element is a fluid-resistance element 104.

The fluid-resistance element 104 is included in the flow path betweenthe first microchannel 24 of the OOC device 10 and the firstoutput-fluid reservoir 106 a. The fluid-resistance element 104 providesa backpressure to the first microfluidic channel 24 by providing apredetermined fluidic resistance to the fluid path associated with thefirst microchannel 24. The fluidic resistance results in a pressure dropor head loss across the fluid-resistance element 104. Beneficially, useof the fluid-resistance element 104 provides for modularity of thesystem 100 because a plurality of fluid-resistance elements 104 havingdifferent fluidic resistances can be used to accommodate differingoperating conditions of the OOC device 10, working-fluid reservoir 102,and output-fluid reservoirs 106 a, 106 b. Additionally, the plurality offluid-resistance elements 104 can be coupled in series and/or parallelto provide for fluidic resistances that are different from thepredetermined fluidic resistances of the individual fluid-resistanceelements 104. The fluid-resistance elements 104 can be, for example, achanneled resistor or a tubular resistor, such as those described inU.S. Provisional Patent Application Ser. No. 62/024,361, filed Jul. 14,2014, which is owned by the assignee of the present application and isincorporated by reference in its entirety.

To describe the functionality of FIG. 3, the fluid travel will bedescribed with respect to an aliquot of the working fluid 108. Analiquot of working fluid 108 flows from the working-fluid reservoir 102to the first microchannel 24 of the OOC device 10 through input line 110at a flow rate of Q₁. Once in the first microchannel 24, the aliquottravels from the input of the first microchannel 24 toward the output ofthe first microchannel 24 across the cells 34. While passing through thefirst microchannel 24, a first portion of the aliquot Q₂ will make it tothe output of the first microchannel 24, and a second portion of thealiquot Q_(t) will travel into the second microchannel 24 through thelayer of cells 34 and barrier 30 (shown best in FIG. 3) due to thepressure gradient ΔP between the first microchannel 24 and the secondmicrochannel 26. The first portion of the aliquot travels from theoutput of the first microchannel 24 to the first output-fluid reservoir106 a through output line 112 a and the first fluid-resistance element104 a. The second portion of the aliquot travels from the output of thesecond microchannel 26 to the second output-fluid reservoir 106 bthrough the output line 112 b.

Once collected by the output-fluid reservoirs 106 a and 106 b, the firstand the second aliquots can be compared as described above to determinethe hydraulic conductivity of the cell layer 34 under dynamic operatingconditions. In the illustrated embodiments, the flow rate of fluidcollected by the second output-fluid reservoir 106 b and fluid in thesecond microchannel 26 is entirely dependent on both the hydraulicconductivity of the barrier (the cell layer 34 and the barrier 30) andthe pressure gradient between the two compartments because the fluid inthe second microchannel 26 is only the fluid that has passed from thefirst microchannel 24 to the second microchannel 26 through the celllayer 34 and barrier 30. Beneficially, because the sum of the firstoutput fluid 114 a and the second output fluid 114 b will equal theworking fluid 108 input to the system, any two of the three values canbe measured or known to determine the hydraulic conductivity of thebarrier. For example, the hydraulic conductivity can be calculated whenthe working fluid 108 flow rate and the first output fluid 114 a flowrate are measured or known, the working fluid 108 flow rate and thesecond output fluid 114 b flow rate are measured or known, or when thefirst output fluid 114 a flow rate and the second output fluid 114 b aremeasured or known. It should be understood that the OOC device 10 can beplaced in the system with only the barrier 30 (i.e. no cellular layer34) to determine the hydraulic conductivity (or resistance) associatedwith only the barrier 30.

In some embodiments, a second flow-determining element is disposed inthe fluid path of the second microchannel 26. This secondflow-determining element provides for independent tuning of the flowrate through the first microchannel and the pressure gradient across thebarrier 30 of the OOC device 10. Beneficially, use of the secondflow-determining element provides for control of conditions in thesecond microchannel 26 and across the barrier 30 without altering theflow rate or other properties of the first microchannel 24. In someembodiments, the second flow-determining element is configured tocontrol the flow rate through the second fluid path. In someembodiments, the second flow-determining element provides a backpressureto increase the pressure within the second microchannel 26, therebyreducing the pressure gradient across the barrier 30.

Referring now to FIG. 5, a schematic representation of a system 200 forquantifying the hydraulic conductivity of biological cell layers 34 isshown, according to aspects of the present invention. Like the system100 of FIG. 4, the system 200 includes the working-fluid reservoir 102,the OOC device 10, the first flow-determining element such as the firstfluid-resistance element 104 a, the second flow-determining element suchas the second fluid-resistance element 104 b, the first output-fluidreservoir 106 a, and the second output-fluid reservoir 106 b. Theworking-fluid reservoir 102, the OOC device 10, the firstfluid-resistance element 104 a, the first output-fluid reservoir 106 a,and the second output-fluid reservoir are the same as or similar tothose described with respect to FIG. 4, above.

The second fluid-resistance element 104 b is included in the flow pathbetween the output of the second microchannel 26 and the secondoutput-fluid reservoir 106 b. The second fluid-resistance element 104 bprovides a backpressure to the fluid in the second microchannel 26 byproviding predetermined fluidic resistance to the fluid path associatedwith the second microchannel 26. The fluidic resistance of the secondfluid-resistance element 104 b is generally lower than the fluidicresistance of the first fluid-resistance element 104 a to allow for apressure gradient ΔP across the cell layers 34 and the barrier 30.Beneficially, the second fluid-resistance element 104 b can be used toalter the effective pressure gradient ΔP across the cell layers 34 andbarrier 30 while the operating pressure and flow rate of the firstmicrochannel 24 remain unaffected by the addition of the secondfluid-resistance element 104 b. Additionally, use of the secondfluid-resistance element 104 b provides for increased modularity of thesystem 200 because the first fluid-resistance element 104 a can beselected to satisfy a first criterion, and the second fluid-resistanceelement 104 b can be selected to satisfy a second criterion. Forexample, the first fluid-resistance element 104 a can be selected toprovide for a predetermined flow rate through the first microchannel 24,and the second fluid-resistance element 104 b to provide for apredetermined pressure drop across the cell layers 34 and the barrier30. As one skilled in the art will appreciate, the selection of thefirst fluid-resistance element 104 a impacts selection of the secondfluid-resistance element 104 b and vice versa.

Flow through the system may be pressure driven or volume driven. In someembodiments, pressure-driven flow is accomplished by pressurizing theworking-fluid reservoir 102 using, for example, a pressurized gas. Insome embodiments, pressure-driven flow is accomplished using gravity by,for example, suspending the working-fluid reservoir 102 a distance abovethe remaining components of the system 100, 200. In some embodiments,volume-driven flow is accomplished using a volumetric pump supplyingfluid to the system 100, 200 at a predetermined rate, for example,supplying fluid to the working-fluid reservoir 102 from an upstreamprocess. In some embodiments, a volumetric pump may be integrated withthe system 100, 200 and coupled to components such as the input line110, or the first or second output lines 112 a, 112 b. Additionally,flow through the system or flow through components of the system can bedetermined, adjusted, and/or controlled using combinations ofpressure-driven flow methods and volume-driven flow methods. Forexample, combinations of volumetric pumps, pressure-driven pumps,pressure regulators, flow-determining elements, and fluid-resistanceelements can be employed to create desired conditions through each flowpath and through the system as a whole.

To monitor fluid pressures in the system, any number of pressure sensorscan be incorporated into the system 100, 200 to measure pressure atpredetermined locations. For example, pressure sensors can beincorporated into fluid lines, reservoirs, the OOC device 10, the firstand second microchannels 24, 26, or other suitable components.

In an example of the system 100 of FIG. 4, the OOC device 10 is apolydimethysiloxane (“PDMS”) microfluidic device. The first and secondmicrochannels 24, 26 have a length of 1.8 cm, a height of 100 and awidth of 400 μm. The first and second microchannels 24, 26 are separatedby a porous barrier 30 having a thickness of about 10 μm and pore sizeshaving a transverse dimension of about 10 μm. The cell layer 34 isformed from human umbilical vein endothelial cells that were grown inthe first microchannel 24 until they reached confluence. The inlet ofthe first microchannel 24 was connected to a 25 cm raised working-fluidreservoir 102 with working fluid 108. The outlet of the firstmicrochannel 24 was connected to a fluid-resistance element 104 having alength of about 1 cm, a height of about 60 μm, and a width of about 100μm. The fluid-resistance element 104 provided a fluidic resistance thatwas more than 10 times higher than the resistance of the firstmicrochannel 24 of the OOC device 10. In this example, the working fluid108 exiting the working-fluid reservoir 102 had a pressure of about 25cm H₂O, the fluid exiting the output of the first microchannel 24 of theOOC device 10 had a pressure of about 24.8 cm H₂O, and the fluid exitingthe fluid-resistance element 104 had a pressure of about 0.1 cm H₂O.

In an example of the system 200 of FIG. 5, a second fluid-resistanceelement 104 b was added to the above-described example system. In thisexample, the working fluid 108 exiting the working-fluid reservoir 102had a pressure of about 25 cm H₂O. The fluid exiting the output of thefirst microchannel 24 of the OOC device 10 had a pressure of about 24.8cm H₂O, the fluid exiting the first fluid-resistance element 104 a had apressure of about 0.1 cm H₂O, the fluid exiting the second microfluidicchannel 26 had a pressure of about 10 cm H₂O, and the fluid exiting thesecond fluid-resistance element 104 b had a pressure of about 0.1 cmH₂O.

Systems and methods in accordance with embodiments of the presentinvention can also be used to more accurately measure the dynamicpermeability of biological layers. Permeability coefficients are therate of diffusive (or in the case of bioactive molecules that areactively transported by tissues, quasi-diffusive) transport from onecompartment to the other. The permeability coefficients are normalizedfor the steepness of the concentration gradient between the twocompartments and for the surface area between the two compartments. Thestandard unit is generally expressed in terms of distance over time,such as m/s, cm/min, or cm/s. Because diffusive or quasi-diffusivetransfer is generally much slower than convective transfer, evenrelatively small amounts of convective transfer can make it difficult,if not impossible, to accurately measure permeability coefficients.

In some embodiments for quantifying the dynamic permeability ofbiological cell layers, fluid is flowed through both the first and thesecond microchannels 24, 26. The fluid flowing through the firstmicrochannel 24 is usually different from the fluid in the secondmicrochannel 26. The fluids may be completely different compositions ormay share one or more components. The pressure drop across the barrierand cell layer(s) is kept as close to zero as possible such thatconvective transfer is minimized and the transfer of compounds betweenthe first microchannel and the second microchannel is due primarily todiffusive or quasi-diffusive transport through the cell layer. Fluidicresistance elements are disposed downstream of each microchannel of themicrofluidic device to minimize the detrimental effects on flow andpressure within the microchannels, which may be caused by blockages suchas small pieces of dirt, clumps of tissue, or small air bubbles thatdevelop in the fluid path through the course of an experiment.

FIG. 6 illustrates a schematic representation of a system 600 forquantifying the dynamic permeability of biological cell layers 34,according to aspects of the present invention. The system 600 includes amicrofluidic device 10, a first working-fluid reservoir 102 a, a firstfluid-resistance element 104 a, a first output-fluid reservoir 106 a, asecond working-fluid reservoir 102 b, a second fluid-resistance element104 b, and a second output-fluid reservoir 106 b. A first input line 110a and a second input line 110 b fluidically couple the firstworking-fluid reservoir 102 a and the second working-fluid reservoir 102b to the respective first microchannel 24 and second microchannel 26 ofthe microfluidic device 10. Output lines 112 a,b fluidically couple thefirst and the second microchannels 24, 26 to the respective firstfluid-resistance element 104 a and second fluid-resistance element 104b, and the respective first and second output-fluid reservoir 106 a,b. Afirst fluid path is defined by the first working fluid reservoir 102 a,the first input line 110 a, the first microchannel 24, the first outputline 112 a, the first fluid-resistance element 104 a, and the firstoutput-fluid reservoir 106 a. Similarly, a second fluid path is definedby the second working fluid reservoir 102 b, the second input line 110b, the second microchannel 26, the second output line 112 b, the secondfluid-resistance element 104 b, and the second output-fluid reservoir106 b.

The first working-fluid reservoir 102 a includes a first working fluid108 a therein that is flowed to the first output-fluid reservoir 106 athrough the first fluid path. The second working-fluid reservoir 102 bincludes a second working fluid 108 b therein that is flowed to thesecond output-fluid reservoir 106 b through the second fluid path. Asthe first working fluid 108 a or the second working fluid 108 b travelsacross the cell layer 34, one or more compounds are transferred betweenthe first working fluid 108 a and the second working fluid 108 b toproduce the first output fluid 114 a and the second output fluid 114 b,respectively. Characteristics of one or both of the first and the secondoutput fluids 114 a,b can be analyzed to determine the dynamicpermeability through the barrier and the cell layer.

FIG. 7 illustrates a flow schematic of the system of FIG. 6. Theschematic includes the first channel 24, the second channel 26, theporous barrier 30, the first fluid-resistance element 104 a, and thesecond fluid-resistance element 104 b. The first microchannel 24 has afirst nominal resistance R_(channel-1), and the second channel 26 has asecond nominal resistance R_(channel-2). The nominal resistancesR_(channel-1), R_(channel-2) are the resistances of the microchannels24, 26 when no blockages are present. The first fluid-resistance element104 a has a first fluidic resistance R_(resistor-1), and the secondfluid-resistance element 104 b has a second fluidic resistanceR_(resistor-2).

The first and second fluidic resistances R_(resistor-1), R_(resistor-2)are substantially larger than the nominal resistances R_(channel-1),R_(channel-2) of the respective microchannel 24, 26 to dampen orminimize the pressure differential across the barrier and associatedcell layer(s). For example, in some embodiments, the first and secondfluidic resistances R_(resistor-1), R_(resistor-2) are between about 10times and about 40 times greater than the nominal fluid resistancesR_(channel-1), R_(channel-2) of the respective microchannel 24, 26. Insome embodiments, the first and second fluidic resistancesR_(resistor-1), R_(resistor-2) are between about 40 times and about 100times greater than the nominal fluid resistances R_(channel-1),R_(channel-2) of the respective microchannel 24, 26. In someembodiments, the first and second fluidic resistances R_(resistor-1),R_(resistor-2) are more than about 100 times greater than the nominalfluid resistances R_(channel-1), R_(channel-2) of the respectivemicrochannel 24, 26. Because virtually all pressure in the system isneeded to overcome the fluid-resistance elements 104 a,b, the pressuredistribution in the first and second microchannels 24, 26 is nearlyuniform. This is true even if there are differences in nominalresistances R_(channel-1), R_(channel-2) of the microchannels 24, 26.Convective flow across the barrier 30 is minimized because pressuredifferences between the two microchannels 24, 26 are insignificant inthe context of the entire system 600.

In summary, the difference in resistances between the fluid-resistanceelements 104 a,b and the microchannels 24, 26 provides for a negligiblepressure drop across the barrier 30 and inhibits convective transferbetween the first and second working fluids 108 a,b, even when ablockage occurs. The difference in resistances can also provide for anegligible pressure drop across the barrier 30 even when there aredifferences in the nominal resistances R_(channel-1), R_(channel-2) ofthe microchannels 24, 26.

By way of example, flow through systems with and withoutfluid-resistance elements 104 a,b will now be described and compared. Inthese examples, the nominal resistances R_(channel-1), R_(channel-2) ofthe microchannels are equal. The first input flow rate {dot over(V)}_(in-1) and second input flow rate {dot over (V)}_(in-2) are each 2μL/min. Under ideal conditions with no pressure differential across thebarrier, the convective flow rate {dot over (V)}_(membrane) is 0 μL/min,and the first output flow rate {dot over (V)}_(out-1) and the secondoutput flow rate {dot over (V)}_(out-2) are each 2 μL/min. But undermore realistic conditions, there is some pressure differential acrossthe barrier because the resistances R_(channel-1), R_(channel-2) of themicrochannels are different due to, for example, differences in flowchannel geometry caused by differences in the geometry of themicrochannel, present or size of tissue within the microchannel, or aminor obstruction such as small pieces of debris, clumps of tissue, orsmall air bubbles within the microchannel. For example, an accumulationof air bubbles can make the actual resistance of the second microchannel26 four times its nominal resistance R_(channel-2). This causes theresistance of the second fluid path to be four times the resistance ofthe first fluid path. The imbalance of resistances increases thepressure differential across the barrier 30 and increases the convectiveflow rate {dot over (V)}_(membrane) across the barrier 30. In thisexample, the convective flow rate {dot over (V)}_(membrane) increasesfrom about 0 μL/min to about 1.2 μL/min. This convective transferoverwhelms any diffusive or quasi-diffusive transfer between the firstand the second working fluids 108 a,b and causes inaccurate or incorrectcharacterization of the dynamic permeability of the cell layer 34.

To overcome the problems with diffusion measurements associated withincreases in resistance (e.g., due to obstructions or differentgeometries), fluid-resistance elements 104 a,b are added to each of thefluid paths. The addition of the fluid-resistance elements 104 a,bsignificantly inhibits convective transfer between the first and secondworking fluids 108 a,b by creating a larger pressure within eachmicrochannel. In this example, the first and second fluidic resistancesR_(resistor-1), R_(resistor-2) are 40 times the nominal resistancesR_(channel-1), R_(channel-2) of the microchannels 24, 26. Thus, each ofthe unobstructed fluid paths has a total resistance of 41 times thenominal resistances R_(channel-1), R_(channel-2). Because the overallresistance of each fluid path is relatively high, an obstruction ineither fluid path will have a much lower effect on the overallresistance of the fluid path. For example, an accumulation of airbubbles in the second microchannel 26 may make the actual resistance ofthe second microchannel 26 four times the nominal resistanceR_(channel-2), but the overall resistance of the fluid path would onlybe about 10% greater than the resistance of the first fluid path (e.g.,45 times the nominal resistances, R_(channel-1), R_(channel-2)). Thisdampens the increased pressure differential across the barrier 30 andinhibits convective flow across the barrier 30 and associated celllayer(s) 34. In this example, the convective flow rate {dot over(V)}_(membrane) increases from about 0 μL/min to only about 0.07 μL/min.Because the convective transfer remains low, diffusive orquasi-diffusive transfer between the first and the second working fluids108 a,b can still be measured. Beneficially, any convective transferthat does occur across the barrier 30 and cell layer(s) 34 can beaccounted for because the flow rates of the first and second workingfluid 108 a,b into the system are generally known, and the convectiveflow is low enough so as to not overwhelm the measurements. In someembodiments, the increased accuracy is achieved by subtracting anyanalyte transported through convective transfer from the measured amountof analyte. This further increases the accuracy of characterizing thedynamic permeability of the cell layer(s) 34 within the system.Additionally, the accuracy of the measured permeability can be increasedby removing the effects of known components known components such as thebarrier 30 or additional cell layers 34. More specifically, the measuredpermeability can be corrected using the following equation:

$P_{Layer} = {P_{Total} - \frac{1}{\frac{1}{P_{Total}} - \frac{1}{P_{Knowns}}}}$

-   Where P_(Total) is the measured permeability, P_(Knowns) is the    permeability of known components such as the barrier and known    layers, and P_(Layer) is the permeability of the unknown layer or    layers.

While the microchannels 24, 26 of the above-described systems 100, 200,600 have had a backpressure applied by fluid-resistance elements 104,the output-fluid reservoirs 106 a, 106 b can be used to provide,supplement, or adjust the backpressure applied to the microchannels 24,26. For example, in system 200, the second fluid-resistance element 104b providing about 10 cm H₂O backpressure. This 10 cm H₂O backpressurecan alternatively be provided by removing the second fluid-resistanceelement 104 b, and raising the second output-fluid reservoir 106 b by 10centimeters from its original position. In yet another alternative, thesecond fluid-resistance element 104 b can provide 5 cm H₂O backpressure,and the second output-fluid reservoir 106 b can be raised 5 centimetersfrom its original position to supplement the backpressure provided bythe second fluid-resistance element 104 b, thus providing a totalbackpressure of 10 cm H₂O.

While the above-described systems 100, 200, 600 have been described asemploying an OOC device 10 having two microchannels 24, 26, it iscontemplated that OOC devices 10 having three or more microchannels.Barriers 30 are disposed between adjacent microchannels of the OOCdevice 10 to allow for diffusion between the adjacent microchannels.Depending on the application, the barrier 30 may have a porosity topermit the migration of cells, particulates, media, proteins, and/orchemicals between the adjacent microchannels. Moreover, the barriers mayhave different porosities. For example, a barrier between a first and asecond microchannel may have a different porosity than a barrier betweenthe second and a third microchannel. The microchannels may be arrangedin any suitable fashion. In some embodiments, a high-pressure channel isdisposed between two lower pressure channels. In some embodiments, ahigh-pressure channel is disposed adjacent a moderate-pressure channel,which is disposed adjacent a low-pressure channel in a “cascadingfashion.” It is contemplated that various combinations of pressures andcell layers may be used on OOC devices 10 having three or moremicrochannels to provide in-vitro modeling of a variety of conditionsthat are otherwise impractical or impossible to replicate (e.g.,exploration of how various backpressures affect transfer through complexsystems, differences in selective permeation between a first cell layerand a second cell layer, etc.).

While the above-described systems 100, 200, 600 have been discussed withrespect to the fluid-resistance elements 104 and output-fluid reservoirs106 a, 106 b providing a constant backpressure, the systems 100, 200 canhave a dynamic fluidic resistance that varies or is selectively variedover time. For example, the fluidic resistance provided by thefluid-resistance elements can be a dynamic fluidic resistance using anair-filled tube that is sealed. In this example, the fluid wouldcompress the air in the tube and the backpressure would increase untilthe pressure of air approximately matches the pressure in the respectivemicrochannel. As the backpressure increases, flow through the respectivemicrochannel would decrease, creating dynamic fluctuations in the systemthat can be used to more accurately simulate conditions in-vivo. Theair-filled tube can be included in, for example, the firstfluid-resistance element 104 a, the second fluid-resistance element 104b, or both. In another example, backpressure is provided by anair-filled tube that is open and extends to a predetermined height. Thispredetermined height can correspond to a maximum desired backpressure,or to a point that is higher than pressure on the remainder of thesystem. In this example, the fluid would fill the air-filled tube andthe backpressure would increase based on the height of the fluid in thetube. Moreover, it is contemplated that fluid-resistance elements 104can be adjusted or altered to increase or decrease fluidic resistance.

While the above-described systems 100, 200, 600 have been discussed withrespect to the fluid-resistance elements 104 being disposed downstreamof the microfluidic device 10, it is contemplated that embodiments ofthe present invention may include fluid-resistance elements 104 beingdisposed upstream of the microfluidic device 10. Further, whileembodiments of the above-described systems 600 have been discussed withrespect to measuring dynamic permeability, it is contemplated thatsystems and methods in accord with the present disclosure may be used tomeasure more general permeability of the cell layers.

Microfluidic devices for cell culture typically need to be perfused withfluid media at an extremely low flow rate, such as between 30 μL/hr and5 mL/hr. Moreover, in some experiments, these flow rates must beconsistent for several weeks. According to existing approaches fordevice interconnection, fluidic (microfluidic and/or non-microfluidic)devices are typically interconnected using tubing and valves thatconnect the output of one device to the input of another. However, theuse of tubing and valves presents some disadvantages, such as the needto limit accumulation of media within the system over the course of theexperiment. Beneficially, the use of fluid-resistance elements alsoallows for systems in accord with the present invention to beincorporated into larger systems regardless of the operating conditionsof the larger system as flow rates and other properties can be selectedand controlled to fit the required operating conditions of the specificsystem while maintaining a desired operating pressures across themicrochannels 24, 26, operating pressure gradient ΔP across the barrier30, and flow rates {dot over (V)}_(in-1), {dot over (V)}_(in-2), {dotover (V)}_(out-1), {dot over (V)}_(out-2) through the system.

Referring now to FIG. 8, a representation of a system 800 forquantifying fluid transport across biological cell layers is shownaccording to aspects of the present invention. The system 800 includes aworking-fluid reservoir 802 and an OOC device 10. As shown best in FIG.1, the OOC device 10 includes a first fluid inlet 14 a, a first fluidoutlet 14 b, a second fluid inlet 16 a, and a second fluid outlet 16 b.A first fluid line 804 a extends between the first fluid inlet 14 a andthe working-fluid reservoir 802. A second fluid line 804 b extendsbetween the first fluid outlet 14 b and the working-fluid reservoir 802.The second fluid inlet 16 a is plugged such that fluid does notgenerally flow through the second fluid inlet 16 a. A third fluid line806 extends from the second fluid outlet 16 b.

When in use, the first fluid line 804 a, the first microchannel 24, thesecond fluid line 804 b, and the working-fluid reservoir 802 form afirst fluid path. The fluid path is loaded with working fluid containedin the working-fluid reservoir 802 such that any movement of fluid fromthe first microchannel 24 to the second microchannel 26 draws workingfluid from the working-fluid reservoir toward the first microchannel 24.The third fluid line 806 is suspended at generally the same height asthe level of working fluid in the working fluid reservoir 802 such thatthe pressure gradient across the barrier 30 is generally zero. In thisway, mass transfer of the working fluid across the barrier 30, such aspressure-driven flow, is inhibited. Accordingly, hydrostatic headpressure is eliminated such that fluid flow from the first microchannelto the second microchannel is due to the cell layer. For example,pumping activity of cells within the cell layer will transport workingfluid from the first microchannel 24 to the second microchannel 26. Thisflow can be monitored by measuring fluid flow through the third fluidline 806. A marker within the third fluid line 806 can be used tomeasure fluid flow by, for example, monitoring displacement of themarker over time. The marker can include, for example, the leading edgeof the working fluid or a gas bubble within the third fluid line.Additionally or alternatively, the weight or volume of the working fluidwithin the third fluid line 806 can be measured to determine the flowrate of working fluid across the membrane.

For purposes of the present detailed description, the singular includesthe plural and vice versa (unless specifically disclaimed); the words“and” and “or” shall be both conjunctive and disjunctive; the word “all”means “any and all”; the word “any” means “any and all”; and the word“including” means “including without limitation.” Additionally, thesingular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise.

While the above detailed description has described particularembodiments with reference to microfluidic components, it iscontemplated that the above-described concepts are applicable to largersystems. These larger systems include, for example, millifluidic andfluidic systems.

While the present invention has been described with reference to one ormore particular embodiments, those skilled in the art will recognizethat many changes may be made thereto without departing from the spiritand scope of the present invention. Each of these embodiments andobvious variations thereof is contemplated as falling within the spiritand scope of the invention. It is also contemplated that additionalembodiments according to aspects of the present invention may combineany number of features from any of the embodiments described herein.

What is claimed is:
 1. A method for measuring hydraulic conductivityassociated with cells, the method comprising: flowing a working fluidthrough a first microchannel at a first flow rate along a layer ofcells, the layer of cells being disposed on a barrier, the first flowrate causing a first shear stress on the layer of cells; applying afirst pressure to the working fluid in the first microchannel to cause aportion of the working fluid to travel to a second microchannel throughthe layer of cells and the barrier; and calculating a hydraulicconductivity of the layer of cells.
 2. The method of claim 1, whereinsome of the portion of the working fluid, that travels through the layerof cells and the barrier, exits the second microchannel.
 3. The methodof claim 2, further comprising collecting the working fluid exiting thesecond microchannel.
 4. The method of claim 3, wherein said calculatingis based on said pressure and said collected working fluid exiting thesecond microchannel.
 5. The method of claim 1, further comprisingperforming an image-gathering technique on the layer of cells during theflowing and applying pressure steps.
 6. The method of claim 2, furthercomprising monitoring a marker associated with the fluid that travelsthrough the layer of cells and the barrier and that exits the secondmicrochannel.
 7. The method of claim 6, wherein said marker comprisesthe leading edge of the fluid that exist the second microchannel.
 8. Themethod of claim 7, wherein the leading edge is monitored in a fluid linecoupled to said second microchannel.
 9. The method of claim 1, furthercomprising applying a fluidic resistance to the working fluid using afirst fluid-resistance element.
 10. The method of claim 9, wherein thefluidic resistance is configured to cause the applied pressure to begenerally constant pressure along the layer of cells.
 11. The method ofclaim 1, wherein said first microchannel is fluidically coupled to aworking fluid reservoir containing working fluid.
 12. The method ofclaim 11, wherein said applying a pressure comprises raising the workingfluid reservoir such that said working fluid flows by gravity.
 13. Themethod of claim 1, wherein pressure is applied while flowing saidworking fluid through said first microchannel.
 14. A method formeasuring hydraulic conductivity associated with cells, the methodcomprising: moving a working fluid through a first microchannel of amicrofluidic device, the microfluidic device including a secondmicrochannel and a barrier located at an interface region between thefirst microchannel and the second microchannel, the barrier including afirst side facing toward the first microchannel and having the cellsadhered thereto; measuring a portion of the working fluid that migratesthrough the cell layer and the barrier and exits the secondmicrochannel; and determining the hydraulic conductivity of the cells.15. The method of claim 14, wherein the pressure gradient across thebarrier is zero.
 16. The method of claim 14, wherein said portion thatmigrates through the cell layer and the barrier moves by an activetransport process.
 17. The method of claim 14, wherein said measuringcomprises monitoring a marker associated with the working fluid thatexits the second microchannel.
 18. The method of claim 17, wherein saidmarker comprises the leading edge of the portion of the working fluidthat exits the second microchannel.
 19. The method of claim 18, whereinthe leading edge is monitored in a fluid line coupled to said secondmicrochannel.
 20. The method of claim 14, wherein said determiningcomprises determining the flow rate of the portion of the working fluidthat migrates through the barrier.
 21. The method of claim 14, furtherincluding applying a fluidic resistance along a fluid path associatedwith the first microchannel.
 22. The method of claim 21, furtherincluding applying a pressure to the working fluid within the firstmicrochannel to create a first pressure differential between the firstmicrochannel and the second microchannel.
 23. The method of claim 14,wherein a fluid path associated with the first microchannel includes afluidic resistor at a location downstream of the first microchannel toensure a substantially constant pressure gradient along the cells on thebarrier.
 24. A method for measuring hydraulic conductivity associatedwith cells, the method comprising: moving a working fluid through afirst microchannel of a microfluidic device, the microfluidic deviceincluding a second microchannel and a barrier located at an interfaceregion between the first microchannel and the second microchannel, thebarrier including a first side facing toward the first microchannel andhaving the cells adhered thereto; monitoring a marker associated with aportion of the working fluid that migrates through the cell layer andthe barrier and exits the second microchannel; and determining thehydraulic conductivity of the cells.
 25. The method of claim 24, whereinthe marker is monitored in a fluid line coupled to said secondmicrochannel.